Pharmacological agents affect important physiological functions, and mutations that alter the drug response are one way to find genes that mediate these functions. Our lab has been isolating and characterizing mutants of the fruit fly, Drosophila melanogaster, that have an altered response to volatile general anesthetics. In the past year, much of the work in the lab has focused on exploiting the molecular identification of two of these genes in order to provide insight into their normal function. The mushroom body defect gene. Mutations in this gene were originally isolated because they displayed gross anatomical defects in a prominent structure of the central brain of Drosophila, the mushroom body. Compared to other central brain mutants, alleles of the mushroom body defect (mud) gene stood out as particulary sensitive to the effects of halothane on a reflex circuit. The mutant phenotype also indicated an important role for the gene in development of the nervous system, especially in the regulation of neuroblast cell division. As reported before, in a collaborative effort our lab identified the gene at the molecular level. We showed that the gene, which is predicted to encode a large polypeptide without obvious vertebrate homologs, is expressed primarily during embryogenesis but also during subsequent development in a sexually dimorphic manner. During the past year, we have made substantial progress on two fronts. First, we enlarged the list of sequenced alleles from 1 to 4, and we used immunoblotting to determine the degree to which these mutations disrupt gene function. In brief, the sequence of all the mutations predict truncation of the ORF and all of them severely depress protein expression. This showed for the first time that the existing mutations are strong hypomorphs; the mutant phenotype thus reflects the principal functions of the gene. Second, antibodies were used to determine the way Mud protein is distributed within the cell. This was undertaken because the conceptual sequence of the mud ORF provided few strong clues into the functional nature of the gene product. In adults, the protein is present at highest concentration in the gonad, a finding consistent with female sterility of the mutants; accordingly, most of our studies focused on this tissue. Although little or no gene product can be found in somatic tissue of the male or female gonad, Mud is abundant in the germ line. In both sexes, the most prominent localization is at the rim of the interphase nucleus of gametes that have already undergone premeitoic divisions. Meiotic divisions are easy to find in the male germ line and here Mud is associated with the periphery of the spindle. There are only a few proteins known to shuttle between the nuclear envelope and the spindle of Drosophila cells. Prominent among them are the small GTPase Ran and its GTPase-activating partner RanGAP, key players in control of the cell cycle. The new results provide us for the first time with a rational basis for exploring how Mud contributes to development. We hypothesize that the protein is involved in Ran-dependent regulation of the cell cyle, either as a chaperone or an effector. According to this view, the degree to which the development of a tissue depends on Mud varies with the degree of redundancy of alternative chaperones/effectors, with developing neuroblasts and developing oocytes particularly at risk. The Dmalpha1U gene. Amongst the first mutations isolated in this laboratory on the basis of altered sensitivity to halothane were variants that, on genetic grounds, were alleles of a locus that had not been studied for over thirty years. The strong alteration of anesthetic sensitivity provoked a determined effort by us to identify the locus at the molecular level. As reported before, we found that the har38 and har85 mutations map to Dmalpha1U, a predicted gene (aka CG1517) that encodes an ion channel belonging to the superfamily of voltage-gated sodium and calcium ion channels. Within the superfamily, CG1517 and its orthologs in vertebrates and other metazoans form a distinct family, one with a novel pore signature. Despite interest in this family, nothing was known of the physiological properties of the channel or its biological significance. Within the past year, we have used immunological techniques to establish the anatomical distribution of the Drosophila channel and to determine the severity of the available mutations. Briefly, the channel is widely expressed in synaptic regions of the fly brain and expression is severely depressed by our anesthesia mutations. To further characterize the mutant phenotype, we entered a collaborative effort to characterize the effect of the channel on daily locomotor cycling of Drosophila. Most dramatically, mutant flies have an inversion of relative locomotor activity in light versus dark. The data suggest that the circadian clock continues to run in the mutants but, although the flies are not blind, photic control of locomotion is dysfunctional. This finding suggests a provocative link to human disease: the human ortholog of Dmalpha1U lies in a candidate region for susceptibility to bipolar disorder. Of course, other candidates from this region must also be considered and it is dangerous to extrapolate too far from data on fruit fly behavior. But bipolar disorder is a disease that is frequently associated with altered diurnal behavior; our demonstration that hypomorphic mutations of this gene lead to altered daily locomotor rhythms in the fly should serve to make the human version of the channel gene a primary focus of attention.